Prevalence of Mycoplasma infection in private cases of buffaloes with clinical mastitis was fundamentally goaled. During five years investigation, 1250 primiparous and multiparous dairy buffaloes located in different villages of Assiut and Sohag Governorates, South Egypt were clinically inspected. Eighty-nine cases showed signs of clinical mastitis and their mammary secretions were tested for the presence of Mycoplasma infection using Polymerase Chain Reaction (PCR). The clinically mastitic buffaloes were categorized into peracute (nine cases), acute (n = 36) and subacute/chronic (n = 44) forms. Mycoplasma infection was detected in 20.22 % of the tested buffaloes. All molecularly Mycoplasma positive—buffaloes were multiparous and characterized by recurrent attacks of mastitis. Mycoplasma bovigenitalium, Mycoplasma bovirhinus and Mycoplasma arginini were detected with frequent detection, 58.62 %, 31.03 % and 10.35 %, respectively. All PCR—tested samples were negative to Mycoplasma bovis. Mycoplasma bovigenitalium was a predominant Mycoplasmal mastitis pathogen detected in buffaloes with subacute/chronic (29.54 %) mastitis rather than acute (13.88 %) form. Mycoplasma infection could not be detected in buffaloes with peracute mastitis. Clinical significance of the detected Mycoplasma bovirhinus and Mycoplasma arginini as mastitis pathogen was discussed. It can conclude that Mycoplasma bovigenitalium become occupying a considerable grade as a contagious mastitis pathogen in dairy buffaloes in comparison with previous studies referring to spread of infection. PCR as a detecting tool is a manageable contrivance and is easier than conventional culturing procedure, but it is still precious in our laboratories as a routine test for diagnosis of mastitis particularly in smallholder farms and/or private cases.