Pythiosis, caused by Pythium insidiosum, is a severe infectious disease affecting humans and animals worldwide. There is an urgent need for a simple and rapid detection method for pythiosis, especially in remote areas where this disease is prevalent. To address this, a colorimetric loop-mediated isothermal amplification assay (c-LAMP) using hydroxynaphthol blue dye as a color indicator has been developed. This method utilized a one-step closed-tube system under a single temperature reaction to detect P. insidiosum, minimizing DNA carry-over contamination and eliminating the need for expensive tools. The test result can be easily read through the color change from violet (negative) to sky blue (positive). When tested with DNA samples from P. insidiosum (n = 51) and other fungi (n = 70), c-LAMP showed a detection sensitivity, specificity, and accuracy of 100.0 %, 95.7 %, and 97.5 %, respectively. The assay detection limit was 1 x 10⁻⁵ ng of DNA template, 10,000 times lower than the reference multiplex PCR assay (m-PCR). c-LAMP also showed a faster assay turnaround time, taking only 65 min, as opposed to the 180 min required for m-PCR. This newly established c-LAMP is rapid, cost-effective, and efficient, making it a promising tool for detecting P. insidiosum in resource-limited laboratories.