Background and objectives: this study aims to evaluate the clinical and microbiological effects of a single subgingival administration of a locally delivered antibiotic gel containing piperacillin plus tazobactam and compare it with a slow-release doxycycline (14%) gel and a placebo gel, following subgingival instrumentation (SI) in patients with severe periodontitis. Materials and methods: sixty-four patients diagnosed with stage III-IV periodontitis were enrolled, were randomly assigned into three groups, and were treated additionally with a single subgingival administration of piperacillin plus tazobactam gel (group A); doxycycline gel (group B); and placebo gel (group C). The primary outcome variable was the change in mean probing pocket depth (PPD) 6 months after the intervention. Secondary outcome variables were changes in mean full-mouth bleeding score (FMBS); full-mouth plaque score (FMPS); overall bleeding index (BOP); pocket closure; and clinical attachment level (CAL), along with changes in the numbers of five keystone bacteria: Aggregatibacter actinomycetemcomitans (A.a.), Porphyromonas gingivalis (P.g.), Prevotella intermedia (P.i.), Tannerella forsythia (T.f.), and Treponema denticola (T.d.). Intergroup and intragroup differences were evaluated at 3 and 6 months. Results: at baseline, the three groups were comparable. An improvement in clinical parameters such as PPD, CAL, and BOP between groups was observed at 3 and 6 months, but without statistical significance (p > 0.05). At 6 months, the intragroup analysis showed a significant reduction in clinical parameters. Even though the piperacillin plus tazobactam group showed slightly higher PPD reduction, this was not statistically significant when compared to both control groups. Conclusions: The groups had similar results, and subgingival instrumentation can be executed without adjunctive antimicrobials, reducing the costs for the patient and the working time/load of the professional.