Gingivitis is the initial stage of periodontitis, one of the most common oral diseases and the primary cause of tooth loss. This study aims to evaluate the effect of toothpaste containing 2% zinc citrate on gingival health and the abundance of three bacteria related to gingivitis and periodontitis. Eleven volunteers with the same oral health status were randomly assigned to the treatment (n = 5) and control (n = 6) groups. The control group used fluoride toothpaste, while the treatment group used fluoride toothpaste supplemented with 2% zinc citrate for 3 months. The plaque index, gingival index, and bleeding index were measured at baseline (0 day), 3 weeks, and 3 months. Dental plaque from four areas of the mouth (FDI criteria) was collected at the same timepoints. A total of 132 dental plaque samples were analyzed using quantitative PCR (qPCR) to monitor the abundance of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia. Toothpaste containing 2% zinc citrate significantly lowered the gingival index and reduced gum bleeding but did not affect the plaque index. It also reduced the total abundance of the three bacteria related to gingivitis and periodontitis in dental plaque over a long-term period. Toothpaste with 2% zinc citrate persistently improves gingival health and reduces the presence of gingivitis-associated bacteria in dental plaque. Chinese Clinical Trial Registry (Clinical trial registration no.: ChiCTR1900020592) (09/01/2019).