Introduction Traditional culture-based techniques for assessing infection of the root canal system are difficult to use and prone to error. Real-time assessment of the microbial status of the root canal system using laser fluorescence would help address these limitations. Methods This study evaluated the performance of thin optical fibers of different diameters, with either plain or conically modified ends, connected to a KaVo KEY 3 laser with an inbuilt 655-nm laser fluorescence diagnostic system. Penetration was tested on sectioned extracted teeth. Fluorescence recordings were made ex vivo in the canals of extracted teeth with known periapical pathology. Several endodontic medicaments and irrigants were also tested for autofluorescence. Results The fibers could reach the apical third of the root canal, unless the canals had distal curvatures greater than 15஠Penetration was greater for conical than for plain fibers. Fluorescence readings were significantly higher in infected canals (range, 19-99) than in noninfected canals and sound radicular dentin (range, 2-8). Of the medicaments examined, only tetracycline-based medicaments gave false-positive fluorescence signals. Conclusions Fluorescence analysis of root canals with optical fiber probes has the potential for real-time assessment of the microbial status of the root canal system in clinical practice.