The aim of this study was to clarify whether positive results for prostate-specific antigen (PSA) and acid phosphatase (AP) occur in postmortem swabs from the genito-anal region in males (n = 80; 4 regions) and females (n = 20; 3 regions) and to calculate the positive predictive value (PPV) concerning the presence of spermatozoa. In male subjects, the highest incidence of positive test results was found in urethral swabs (PSA 76%, AP 71%) and the lowest frequencies appeared in perianal and rectal swabs (15–20%). Microscopic evaluation for spermatozoa was positive between 39% in urethral swabs and 1% in rectal swabs. PPV regarding positive identification of spermatozoa was 33.3% for PSA and 31.5% for AP. The combination of both tests yielded a PPV of 38.2%. In female cases, no spermatozoa were identified, and one case was PSA- and AP-positive in perianal swabs. Our findings indicate that PSA and AP tests are of limited value for the postmortem detection of spermatozoa in male subjects.