Avian influenza is one of the major threats to poultry and human health in northern Benin, while mixed-species farming systems increase the risks of viral transmission. The present study estimated the seroprevalence of avian influenza subtypes H9N2 and H5 in indigenous chickens and guinea fowls in the Atacora and Donga regions. A total of 300 birds including 191 indigenous chickens and 109 guinea fowls, from six districts were sampled through a cross-sectional survey using systematic random sampling. Hemagglutination inhibition assay was used to detect antibodies, revealing an overall H9N2 seroprevalence of 41%, with 17.5% of samples testing positive for H5. The seroprevalence of H9N2 was notably higher in guinea fowls (51.81% in Atacora and 52% in Donga) compared to chickens (34.95% in Atacora and 34.83% in Donga). H5 antibodies were found only in guinea fowls in Atacora (46.66%). The study also found that farms with both chickens and guinea fowls had a significantly higher odds ratio for H9N2 positivity (OR = 4.25, p < 0.001) compared to chicken-only farms. The results underscore the importance of mixed-species systems in the transmission of avian influenza, suggesting that targeted surveillance and biosecurity measures are essential for controlling the spread of these viruses.