Health Protection Scotland (HPS) reported an increase in incidence of the illness, which affects vulnerable patients, between April and June.
Rates rose 8% in patients over the age of 65 and 13% among 15 to 64-year-olds.
HPS stressed the nine months to June showed a drop in C. Diff in Scottish hospitals. Levels are also lower than in spring 2012.
Dr Camilla Wiuff, strategic lead for microbiology at HPS, said: "At the moment we do not have an explanation for the rise."
She added the number of C. Diff infections would vary, but there were fewer outbreaks on Scottish wards compared to six or seven years ago.
Tighter control of antibiotics prescribed and a crackdown on hygiene in hospitals may have cut levels of C. Diff in Scottish hospitals.
A total of 425 new cases were recorded between April and the end of June this year. In the same period in 2009 there were more than 1000 new cases reported in the 65-plus age group.