The present study aimed to examine the clinical and pathogenic characteristics, diagnosis and treatment of primary canaliculitis to provide further guidance for its clinical management. The present prospective study enrolled 50 patients (50 eyes) diagnosed with primary canaliculitis between May 2018 and April 2021 at Department of Ophthalmology, Affiliated Wuxi Clinical College of Nantong University, Wuxi, China. The patients' general clinicopathological information, clinical characteristics, microbiological profiles and treatment outcomes were analyzed and summarized. All the patients presented with persistent red eyes and eye discharge. Examination of discharge smears revealed that 96% of patients tested positive for Actinomyces and all smears were negative for fungi. Microbial cultures indicated that 82% of cases were positive for bacteria. A total of 51 bacterial strains were cultured; of these, 27.5% were aerobes, 35.3% were anaerobes and 37.2% were facultative anaerobes. A total of 56.9% of strains were gram-positive and 43.1% were gram-negative. The three most common bacteria, including Streptococcus spp., Capnocytophaga spp. and Propionibacterium, were analyzed. Only 3 cases (6%) of microbial cultures were positive for Actinomyces and all cases were negative for fungi in microbial cultures. Among the 50 cases, 45 were cured with conservative treatment [intracanalicular ointment infiltration (IOI)]. Five patients responded poorly to conservative treatment; however, they were cured with surgical treatment. In the current study, the majority of canaliculitis cases were caused by mixed infections, predominantly Actinomyces. The results revealed that the culture positivity rate of Actinomyces was low; however, the smear staining positivity rate was high. Fungus was smear- and culture-negative in all cases. In conclusion, patients with canaliculitis had a good prognosis after timely diagnosis and treatment.