Background: We estimated the occurrence rate of the booster phenomenon by using an intradermal test with 43 kDa glycoprotein in an endemic area of paracoccidioidomycosis in the central-west region of Brazil. Methods: Individuals who had a negative result on a survey performed by using an intradermal test with 43 kDa glycoprotein in an endemic area of paracoccidioidomycosis underwent a second intradermal test after 10–15 days to determine the presence or absence of the booster phenomenon. Statistical analyses were performed using the Chi-square test, Chi-square for linear trend test, Student’s t test, and binomial test; p\0.05 was considered significant. Results: For the first time, we reported the occurrence of the booster phenomenon to an intradermal reaction caused by 43 kDa glycoprotein at a rate of 5.8–8.4%, depending on the test’s cutoff point. This suggests that a cutoff point should be considered for the booster phenomenon in intradermal tests with 43 kDa glycoprotein: a difference of 6–7 mm between readings according to the first and second tests, depending on the purpose of the evaluation. Conclusion: The results indicate that the prevalence of paracoccidioidal infection in endemic areas is underestimated, as the booster phenomenon has not been considered in epidemiological surveys for this infection.