Oral mucositis is still one of the most painful side effects of chemotherapeutic treatment and a mounting body of evidence suggests a key role for the oral microbiome in mucositis development. However, the underlying mechanisms remain elusive. In this work, we have investigated the interactions between the host, the microbiome, and chemotherapeutic treatments in more detail. The effect of 5-fluorouracil, commonly inducing mucositis, was assessed on a co-culture model that consists of an epithelial cell layer and a biofilm derived from oral microbiota from different types of samples (saliva, buccal swabs and tongue swabs) and donors (healthy individuals and patients suffering from mucositis). After 24 h co-incubation, all oral microbial samples were found to reduce wound healing capacity with 2615% as compared with untreated condition. Compared with saliva and tongue samples, buccal samples were characterized by lower bacterial cell counts and hence higher wound healing capacity. For samples from healthy individuals, an inverse correlation was observed between bacterial cell counts and wound healing capacity, whereas for patients suffering from mucositis no correlation was observed. Moreover, patient-derived samples had a less diverse microbial community and higher abundances of pathogenic genera. No major impact of 5-fluorouracil on wound healing capacity or the composition of the microbiome was seen at physiologically relevant concentrations in the mouth. In conclusion, bacterial cell count is inversely correlated with wound healing capacity, which emphasizes the importance of oral hygiene during oral wound healing in healthy individuals. However, future research on extra measures besides oral hygiene is needed to assure a good wound healing during mucositis, as for patients the bacterial composition seems also crucial. The direct effect of 5-fluorouracil on both the microbiome and wound healing is minimal, pointing to the importance of the host and its immune system in chemotherapy-induced microbial shifts.