The soil fauna of the tropics remains one of the least known components of the biosphere. Long-term monitoring of this fauna is hampered by the lack of taxonomic expertise and funding. These obstacles may potentially be lifted with DNA metabarcoding. To validate this approach, we studied the ants, springtails and termites of 100 paired soil samples from Barro Colorado Island, Panama. The fauna was extracted with Berlese-Tullgren funnels and then either sorted with traditional taxonomy and known, individual DNA barcodes (“traditional samples”) or processed with metabarcoding (“metabarcoding samples”). We detected 49 ant, 37 springtail and 34 termite species with 3.46 million reads of the COI gene, at a mean sequence length of 233 bp. Traditional identification yielded 80, 111 and 15 species of ants, springtails and termites, respectively; 98%, 37% and 100% of these species had a Barcode Index Number (BIN) allowing for direct comparison with metabarcoding. Ants were best surveyed through traditional methods, termites were better detected by metabarcoding, and springtails were equally well detected by both techniques. Species richness was underestimated, and faunal composition was different in metabarcoding samples, mostly because 37% of ant species were not detected. The prevalence of species in metabarcoding samples increased with their abundance in traditional samples, and seasonal shifts in species prevalence and faunal composition were similar between traditional and metabarcoding samples. Probable false positive and negative species records were reasonably low (13–18% of common species). We conclude that metabarcoding of samples extracted with Berlese-Tullgren funnels appear suitable for the long-term monitoring of termites and springtails in tropical rainforests. For ants, metabarcoding schemes should be complemented by additional samples of alates from Malaise or light traps.

Although studies of insect decline have recently dominated headlines worldwide, their interpretation requires caution since for most species, we lack long‐term population baselines. In the tropics, where most insect species thrive, our knowledge is even more limited and so reliable insect assessments must originate from well‐established long‐term monitoring efforts. Combining the extensive monitoring data from the Arthropod Program of the Smithsonian Tropical Research Institute (STRI) on Barro Colorado Island (BCI), Panama, we compare whether known arthropod diversity can be detected through metabarcoding of bulk insect samples obtained through automatic light‐trapping. Our study detected 4402 species based on Barcode Index Numbers (BIN) and detected fine‐scale differences between wet and dry seasons and sampling localities. We further refined our analysis to indicate which families and genera explained seasonal turnover. Using samples collected in parallel, but sorted manually as part of the ongoing arthropod monitoring program, we compared these methods. Out of 538 BINs recovered through manual sorting, there was a 70% overlap with the metabarcoding data; however, it represented 30% of the total BINs detected through metabarcoding. Expecting higher detection through metabarcoding, we also compare the results with the 14 years of sampling in BCI to better understand how well the monitoring program has captured the diversity of focal groups. Our results revealed a ~50% overlap between both methods and similar total catch. Barcode Index Numbers manually detected but not recovered by metabarcoding highlight some of the limitations of molecular detection methods such as primer bias. Contrastingly, BINs detected with metabarcoding, but not recovered by the traditional monitoring scheme, highlight the importance of local and regional barcode reference libraries.