Abstract In recent years, for economical purposes and to improve well-being conditions of cows, several dairy producers have adopted litter made from recycled manure solids (RMS). However, its use raises several safety issues, particularly with respect to mastitis and other infections. We previously observed cases of mastitis caused by K. pneumoniae and K. variicola, and two deadly infections from C. perfringens from cows on RMS. A herd of Holstein cows, distributed in two barns of about 40 lactating cows, was used. RMS was used in one barn, and wood-chip litter (WCL), in the other. RMS was generated by roller presses separation followed by conditioning at 55°C for 24h. Clean and soiled (used) RMS and WCL samples were collected for 15 months. Similarly, material samples before conditioning were also collected, and all samples were analyzed by viability-qPCR. Milk samples from cows with mastitis and somatic cell count data (SCC) were analyzed. Total and living contents of K. pneumoniae, K. variicola, C. perfringens and Klebsiella spp. were higher in the RMS than the WCL (+1.6–4.2 log10, P < 0.05). Composting 24h at 55°C did not affect total and live levels of C. perfringens, K. pneumoniae, and even increased total and live levels of K. variicola (+0.9 log10, P < 0.05). A seasonal effect or the number of cycles recycling the RMS decreased levels of K. pneumoniae in winter. Overall, RMS had no significant effect on SCC, but increased mastitis incidence (+39%, P < 0.05), compared to WCL. Milk analysis revealed that higher levels of K. pneumoniae found in RMS could contribute to the increase in mastitis cases caused by this pathogen. Data reported from this study are not favorable to the use of an RMS conditioned for 24h at 55°C with dairy cows, apropos the microbiological aspect; however, these results must be confirmed by other studies.