Aim To evaluate the bacterial composition of collared peccary semen and foreskin mucosa, and to verify the sensitivity of isolates to antimicrobials used in semen conservation and to Aloe vera gel, which is an alternative external cryoprotectant. Methods and Results Nine foreskin mucosa and ejaculate samples from adult animals were used. Sperm characteristics and bacterial load were evaluated in fresh semen. The preputial mucosa and semen bacterial isolates were identified and tested against five concentrations of each antimicrobial (streptomycin-penicillin and gentamicin) and A. vera gel. Corynebacterium sp. and Staphylococcus sp. were isolated in greater numbers than others in both semen (64 center dot 10 and 20 center dot 51%, respectively) and the foreskin mucosa (60 center dot 60 and 24 center dot 25%, respectively), and ranged from 0 center dot 4 to 21 x 10(5) colony-forming units (CFU) per ml. The average load of Corynebacterium sp. was negatively correlated (P < 0 center dot 05) with the sperm membrane integrity (r = -0 center dot 73055) and curvilinear velocity (r = -0 center dot 69048). Streptomycin-penicillin and gentamicin inhibited most micro-organisms, and A. vera showed lower antimicrobial activity. Conclusion Several Gram-positive bacteria are present in semen and foreskin mucosa of collared peccary, and the benefits of using primarily penicillin-streptomycin and gentamicin antimicrobials in the bacterial control of diluted semen of these animals are strongly indicated. Significance and Impact of the Study This study provides insight into the reproductive microbiota of captive male-collared peccary. This work provides a theoretical basis to assist reproductive biotechnologies for ex situ conservation of the species.