This study investigated the efficiency of a chlorhexidine varnish and an antibiotic paste in suppressing the cultivable microflora of deep dentine cavities in a stepwise excavation procedure. Subsequent to enamel preparation and removal of the central biomass, infected dentine was sampled from the cavity floor. Ten cavities each were either covered with the 1% chlorhexidine- and 1% thymol-containing varnish Cervitec (CE), the demeclocycline hydrocortisone-containing ointment Ledermix (LE) or received no treatment as control (CO). A compomer composite was used as intermediate restorative. Cavities were reassessed after 6 weeks and again dentine samples were microbiologically investigated for total viable counts, mutans streptococci and lactobacilli. After 6 weeks a significant reduction of the total viable counts was observed in the LE group (p = 0.011) compared to the control, whereas no differences were found in the CE group (p > 0.05). Mutans streptococci were rarely recovered at baseline and after 6 weeks. Compared to the CO group counts of lactobacilli were significantly reduced in the CE and LE groups (p < 0.05). Lactobacillus species were frequently recovered at baseline and after 6 weeks of observation. Lactobacillus rhamnosus was the predominant species in all samples investigated. Application of CE or LE resulted in reduced counts of lactobacilli after a period of 6 weeks. Although none of the materials completely eliminated the viable microorganisms, the use of LE was more effective than CE in reducing the total anaerobic microorganisms associated with carious dentine.