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Over the last few years, antimicrobial shampoo therapy has been increasingly used to treat skin infections in order to reduce systemic use of antibiotics. This study was aimed to compare the In vitro bactericidal effect of a black currant oil based shampoo (S1) to a chlorhexidine 4% shampoo (S2) against methicillin-sensitive Staphylococcus pseudintermedius (MSSP), methicillin-resistant Staphylococcus pseudintermedius (MRSP), Staphylococcus aureus (SA), Escherichia coli (EC) and Pseudomonas aeruginosa (PA) isolates. A collection of 50 bacterial strains from skin swabs of dogs with superficial recurrent pyoderma was selected: 10 MSSP, 10 MRSP, 10 SA, 10 EC and 10 PA. The two shampoos were blindly tested in duplicate with a microdilution plate method, with scalar concentrations from 1:2 to 1: 256. The MBC was performed for each dilution. A linear regression was used to detect a statistically significance between the two shampoos. All isolates were completely killed at 1:2 up to 1:16 dilution of the two antiseptic products. At the 1:32 dilution the first bacterial growths were observed, in particular for 2 and 4 strains of MRSP by S1 and S2 respectively. The first lethal dilution for SA was at 1:64 for S1/S2 and only for S2 against SP. No significant difference was observed between the two shampoos according to the results of linear regression significant for: i) MRSP, PA and EC (p < 0.05); ii) MSSP and SA (p < 0.1). This study showed that both black currant oil based shampoo and chlorhexidine 4% shampoo have a similar In vitro bactericidal activity.