Edema disease (ED) of pigs is an enterotoxaemic disease caused by enterotoxaemic Escherichia coli (ETEEC) infection. Antimicrobial therapy for pigs with ED is controversial because it may induce death of sickish piglets. In this study, we investigated the effects in vitro of 7 antimicrobial agents, ampicillin, gentamicin, colistin, bicozamycin, fosfomycin, sulfamethoxazole-trimethoprim and enrofloxacin, on the release and production of shiga toxin (Stx) 2e by ETEEC strains. We found that more Stx 2e accumulated in the bacterial cells than was released into supernatant. Associated with inhibition of cell wall synthesis, the exposure to ampicillin or fosfomycin increased the release of Stx 2e. The production levels of Stx 2e in all antimicrobial-treated cultures were equal to the level in the control or less than in the control. These results suggest that cell wall synthesis inhibitors, such as ampicillin and fosfomycin, may change for the worse in the signs in ETEEC infectious pigs. On the other hand, gentamicin, colistin, bicozamycin and enrofloxacin may be useful for the treatment of pigs with ED.