Antimicrobial peptides (AMPs) are an important component of cutaneous innate immunity. The amount of AMPs secreted in healthy and atopic patients is controversial. To evaluate the antimicrobial effect of skin wash harvested from healthy and atopic dogs, the amount of AMPs (cBD3-like/cBD122, cCath, S100A8/9/12) present in the skin washes, and the correlation between AMP levels and severity of the clinical signs. Fifteen healthy and 15 mildly atopic, privately owned dogs. Skin washes were performed on a 15 cm2 area of nonlesional abdominal skin. The area was rinsed with 2.7 mL of saline using a sterile tube. Commercially available (S100A8/9/12) or in-house (cBD3-like and cCath) canine-specific ELISAs were performed. Skin washes were incubated with different concentrations (5 × 105 to 4 × 103 cfu/mL) of Staphylococcus pseudintermedius and the bacterial density measured after 24 h of incubation. Severity of clinical signs was assessed by the Canine Atopic Dermatitis Extent and Severity Index, 4th version (CADESI-04). Data were analysed statistically and significance level was set as P ≤ 0.05. There was no significant difference in any of the tested AMPs between healthy and atopic dogs. Strong/moderate positive correlations were seen between total and inguinal CADESI-04 (r = 0.69; P = 0.0061) and between AMPs [cBD3-like/cCath (r = 0.69; P = 0.011), S100A8/S100A9 (r = 0.8; P = 0.0016) and S100A9/S100A12 (r = 0.57; P = 0.047)]. Significantly less antimicrobial activity was seen in skin washes from atopic versus healthy dogs (P < 0.05). This study found no difference in AMP secretion levels between healthy and nonlesional atopic canine skin. However, a significantly lower antimicrobial activity was present in the skin washes from atopic compared to healthy skin.