The aim of this study was to evaluate the influence of the antibiotic treatment, including the mode of drugs administration, on bitches' attractiveness to the stud dogs during mating. Moreover, we tried to estimate the possibility of aversive effect of the drug vehicle on the male behavior. In experiment I, four bitches in oestrus without antibiotic treatment (group A), four bitches treated with intravaginal antibiotic (group B) and four bitches treated with intramuscular antibiotic (group C) were presented to four stud dogs. In experiment II, bitches in oestrus (n = 5) were presented to the males (n = 2) before and after the application to the females' vulva the antibiotic carrier--Miglyol 840 (Sasol, Germany). In both experiments the presence of the typical sexual behavior of the males (sniffing, licking the vulva and anal region, mating attempts) was evaluated. In experiment III the reaction of the males to the samples containing oestrual discharge from the bitches untreated and treated with antibiotics was evaluated. In the last part of study the aversion reaction to the samples containing antibiotic and the antibiotic carrier was evaluated. The results of experiments showed that females treated with the antibiotics were less attractive to males than untreated females, regardless of the method of administration. We did not observe adverse effect of the antibiotic carrier but samples from the bitches treated with antibiotics were significantly less attractive to the males. We concluded that the reason for reduced attractiveness of the bitches in oestrus after antibiotic treatment was the changes in semiochemical signal emitted by treated females as a consequence of elimination of the vaginal bacterial flora, which seems to be involved in creation of the typical, recognizable by the stud dogs, oestrual signal but also by the possible covering effect of used drugs.