Background: Pain remains one of the main causes of apprehension during dental procedures. In this context, topical anesthetics play an essential role in reducing discomfort, and new compounds such as amitriptyline and jambu (Acmella oleracea) extract have been investigated as promising alternatives. Drug delivery systems, such as PentravanⓇ , a nanosomal transdermal base, are also of interest; however, their application on oral mucosa has not yet been clinically evaluated. Therefore, this study aimed to compare the anesthetic efficacy of lidocaine (LDC) and prilocaine (PLC) combined with either amitriptyline or jambu, formulated as ointments or incorporated into PentravanⓇ , for topical anesthesia of the buccal and palatal mucosa of maxillary premolars, using pulpal response and puncture sensitivity as outcome measures. Methods: A randomized, blinded, crossover clinical trial was conducted with 40 healthy volunteers who received six formulations: (1) LDC 2.5% + PLC 2.5% (positive control); (2) LDC 2.5% + PLC 2.5% + amitriptyline 5%; (3) LDC 2.5% + PLC 2.5% + jambu extract 20% (7.5% spilanthol); (4) LDC 2.5% + PLC 2.5% + amitriptyline 5% in PentravanⓇ ; (5) LDC 2.5% + PLC 2.5% + jambu extract 20% in PentravanⓇ ; and (6) base ointment without active ingredients (placebo). Pain upon needle puncture was assessed using the visual analog scale, and pulpal anesthesia was evaluated with an electric pulp tester. Results: Formulations 1 and 2 produced the lowest pain scores in the buccal mucosa, with Formulation 1 showing statistically superior results than Formulations 3, 4, 5, and 6 (P < 0.05). Formulation 6 yielded the highest pain scores, while Formulations 2–5 demonstrated intermediate efficacy without significant differences.Formulations 1 and 2 were also the most effective on the palatal mucosa. None of the formulations achieved significant pulpal anesthesia. Adverse effects were mild and self-limiting, with mucosal desquamation observed only with Formulation 2. Conclusion Amitriptyline demonstrated relevant clinical potential, whereas jambu extract showed intermediate efficacy. PentravanⓇ -based formulations did not exhibit clinical superiority. Future studies should focus on optimizing these combined formulations to enhance the efficacy of topical anesthesia in dentistry.