Topical therapy is essential in assisting with the resolution of pyoderma. (i) Evaluate the in vitro efficacy and residual activity of two different hair segments treated with shampoo and mousse against meticillin-sensitive and meticillin-resistant staphylococci; (ii) compare proximal and distal hair portions treated with the products and (iii) describe a new disc diffusion method for assessing residual efficacy. Eleven privately owned, medium-haired dogs. In this randomised, blinded and negatively controlled study, dogs were treated once with a 3% chlorhexidine digluconate-0.5% ophytrium shampoo on the lateral thorax, and the corresponding mousse on the opposite side. Hairs were plucked before treatment, two hours post-treatment, and day (D)2, D4, D7, D10 and D14. Hairs were weighed (0.01 g) and cut (1.0 cm) from the proximal portion, moistened with saline and placed on a sterile diffusion disc to absorb the solution. Proximal and distal hair bundles and diffusion discs were placed onto agar inoculated with an isolate of meticillin-sensitive or meticillin-resistant Staphylococcus pseudintermedius or Staphylococcus schleiferi. Inhibition zones were measured following incubation. Distal hairs had larger (p < 0.001) inhibition zones compared to proximal hairs. Mousse had significant differences (p < 0.05) between time points and locations for both the hair bundles and discs, while shampoo only had a significant difference (p < 0.001) between time points for the hairs. Mousse was effective, and shampoo was only minimally effective in inhibiting bacterial growth in vitro, with the greatest effect occurring at the two hours time point. The distal hair shafts had greater inhibition.