Mycoplasma, solely culturable bacteria with the smallest genome, is an important organism to understand the minimal form of life. Mutagenesis using mutagens is a useful methodology for understanding the essential regions of genomic information. Ultraviolet light and trimethyl psoralen are mutagens known to induce various mutations; the latter is reported to specifically induce deletions in nematodes. However, their mutagenic effects on mycoplasma are not known. Here, we exposed Metamycoplasma salivarium to ultraviolet (UV) light or trimethyl psoralen and UV as mutagens, and analyzed the mutational pattern after several rounds of serial cultivation ranging from 34 to 56 for different lineages. Our results showed that more deletions, but fewer point mutations, were induced with TMP and UV-A than with UV alone, indicating the usefulness of TMP in inducing deletions. In addition, we compared our results with mutational data from other studies, which suggested that both TMP-UVA and UV exposure induced point mutations that were highly biased toward C to T and G to A transitions. These data provide useful basic knowledge for mutational studies on M. salivarium.