We evaluated the influence of abiotic factors on antagonistic activity of Actinobacillus actinomycetemcomitans strains isolated from periodontal pockets and two reference strains (ATCC 29523 and FDC Y4). Antagonistic assays were performed by the overlayer method on tryptic soy agar (TSA), brain heart infusion agar, thioglycollate agar and brucella agar, added with yeast extract and supplemented (S) or not with L-cystine and sodium bicarbonate. Iso-, auto-, and heteroantagonism against a wide range of indicator strains were assayed. The influence of incubation atmosphere (anaerobic chamber, anaerobic and candle jars) and pH (5.0 to 11.0) was also evaluated. Autoantagonism was not observed. TSA-S was shown to be the most adequate medium for antagonistic activity expression. The widest spectrum of heteroantagonistic activity was also observed on TSA-S. The incubation atmosphere affected only the isoantagonistic activity expression. Only at pH 8.0 did A. actinomycetemcomitans express bacteriocinogenic activity. The lack of standardized methodology to detect antagonistic activity can lead to discrepant results and can make data difficult to be compared. This study provides information on abiotic factors that influence bacteriocinogenic activity expression and suggests adequate culture conditions for testing A. actinomycetemcomitans bacteriocin production, contributing to the establishment of a reproducible and reliable methodology.