Dental plaque is the main cause leading to the dental caries and periodontal diseases. The main purpose of this study was to test the efficacy of oral spray containing the antimicrobial peptide P-113 on the reduction of oral bacteria number and dental plaque formation in a randomized clinical assessment. This study was divided into two parts. In Part A, we investigated the user experiences with the P-113 containing oral spray. In part B, 14 subjects in the experimental group used the P-113-containing oral spray, while 14 subjects in the control group used a placebo without the P-113 in a 4-week clinical trial. Participants were asked to use the P-113-containing oral spray or placebo 3 times per day and 5 times per use. Moreover, 3 check-ups and 2 washouts were carried out to evaluate the DMFT score, dental plaque weight, dental plaque index, and gingival index. In part A, up to 91.8% of the subjects in the experimental group were satisfied with the use of the P-113-containing oral spray. In part B, based on our PacBio SMRT sequencing platform and DADA2 analysis, the numbers of Streptococcus and Porphyromonas in the experimental group were lower than those in the control group. In addition, decreased dental plaque weight, dental plaque index, and gingival index were all observed in the experimental group. The P-113-containing oral spray has the potential to reduce the dental caries and periodontal disease-related bacteria and to control the dental plaque formation.