Rates of C. diff infections climbed by 19% in the over 65s and 35% in younger adults during last summer.
Health Protection Scotland said it was not unusual for C. diff rates to peak from July to September, but said hygiene failures and inappropriate use of antibiotics could be behind the rise.
Scotland has made big improvements in controlling hospital acquired infections in the last 10 years. The number of people contracting C. diff infections is down 80% since records began in 2006 and cases of superbug MRSA reached the lowest level since 2005 last summer with 29 cases recorded.
Health Secretary Alex Neil said: "I am delighted to see that our efforts to tackle hospital acquired infections have seen levels of MRSA fall once again. These results show what can be achieved by supporting NHS staff to embed good practice."
However, from July to September last year, there were 351 new cases of C. diff in over 65s, up from 314 the previous quarter, and 152 new cases in the 15 to 64 category, up from 112.
Scottish Labour's health spokesman Neil Findlay said: "We already know our health care professionals are under increasing pressure as a result of SNP cuts to nursing staff and bed numbers so this situation must be carefully monitored and support provided to prevent the number of people contracting C. diff in hospitals from rising further."
The Scottish Government is introducing new facilitators to work with frontline staff to tackle infections.