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Dentine caries has a microbiological aetiology whilst the diagnosis relies on clinical signs. This study examined relationships between the Locations, Colours, Textures, Sizes, Perceived Treatment Needs, Cavitations and microbiological characteristics of Primary Root Caries. The relationships between some microflora of Primary Root Caries deemed to require restoration and the overlying plaque as well as the effects of a Chemotherapeutic agent on some microflora of lesions were also studied. In all, 610 lesions in 303 patients were investigated. 447 lesions in 169 patients were examined in the main study. The Locations of lesions were related to the gingival margins; Colours were designated: Black, Yellow, Light Brown or Dark Brown; Textures were recorded as Hard, Leathery or Soft, Sizes as products of Heights and Widths, and Cavitations as the greatest loss of surface contour. The total numbers of colony forming units ; Gram-positive pleomorphic rods; Mutans streptococci; Lactobacilli; and Yeasts expressed as Log1p as well as the proportions and Frequency of isolation, in each dentine biopsy were determined, eg - 90.9 percent of Soft but only 3.3 percent of Hard lesions were <1 mm from gingivae (P < 0.001); - 68.6 percent of Soft but only 6.7 percent of Hard lesions were sized >7 mm2 (P < 0.01); - more cavitated lesions were larger (P < 0.01); - higher total numbers, proportions and frequencies of isolation of Mutans streptococci and Lactobacilli were in Soft and Leathery than in*Hard lesions (P < 0.01); - the mean total numbers of colony forming units, Mutans streptococci, Lactobacilli and Gram-positive pleomorphic rods were less in each group of lesions with a reduced Perceived Treatment Need (P < 0.01); - lesions deemed to require restoration most frequently contained Yeasts (P < 0.01); - the most reliable indicators of microbiological activity were Texture and Location rather than Colour; From 81 Primary Root Caries lesions deemed to require restoration in 52 patients, not amongst the 169 in the larger study, higher proportions of Gram-positive pleomorphic rods (P < 0.001) and Lactobacilli (P < 0.01) were in carious dentine than in the associated plaque, indicating that precision in sampling is paramount. 42 of 82 lesions deemed to require restoration in another 82 patients, were coated with a varnish containing 1 weight (wt) percent chlorhexidine and 1 wt percent thymol and after 24 hours these and the lesions not varnished were biopsied. The varnish significantly reduced the numbers of micro-organisms (total colony forming units, P<0.001; Mutans streptococci, Lactobacilli and Yeasts, P<0.01). These studies will help clinicians and epidemiologists to diagnose the levels of activity in Primary Root Caries and to indicate how Chemotherapy rather than the removal of carious dentine might be developed as a preferred strategy for its management.