The mentum deformity incidence in Chironomus plumosus larvae to assess the environmental contamination level in Lake Trasimeno, Central Italy, was investigated. The survey lasted from May 2018 to August 2019. Fifty-one samplings were carried out: 34 in the littoral zone and 17 in the central zone. The deformity assessment was based on 737 and 2767 larval specimens of C. plumosus collected from the littoral and central zones, respectively. Comparison of the larval morphometric variables between normal and deformed specimens highlighted that the deformities did not cause alterations of the larval growth. The deformity incidence amounted to 7.22% in the whole Trasimeno’s ecosystem, reaching 8.28% in the littoral zone and 6.94% in the central zone. Among the different seasonal cohorts, the spring cohort had overall the highest deformity value (11.41%). The deformity type assessment protocol highlighted that the most common deformity type was “round/filed teeth” (64%). The results of this 2018–2019 survey revealed a low deformity incidence, within the background range of relatively low-impacted freshwaters. Comparison with previous investigations (2000–2010) of the same habitat showed a clear decrease of the deformity incidence. This study further contributes to the evaluation of the mentum deformity in chironomids that represent an indicator endpoint of the anthropogenic contamination level in freshwaters.