Background and Objectives: Low back pain (LBP) represents a major cause of disability worldwide, with a significant impact on quality of life and functional capacity. Standard physiotherapy is widely used for LBP, whereas comparative short-term evidence on balneotherapy and prolotherapy remains limited. This study aimed to compare clinical outcomes across therapeutic approaches in patients with LBP. Materials and Methods: This non-randomized, three-group interventional cohort study included adult patients diagnosed with LBP lasting more than three months and presenting a baseline Visual Analog Scale (VAS) score ≥ 4. 84 patients were allocated to one of three treatment groups: standard physiotherapy (CG) (25 patients), balneotherapy plus physiotherapy (BG) (28 patients), or prolotherapy (PG) (31 patients). Outcome measures included pain intensity, functional disability, lumbar mobility, quality of life, and psychological status. This clinical study has been officially registered on ClinicalTrials.gov under the identifier NCT07399145. Results: Statistical analyses were performed to assess within- and between-group differences over time. At the 4-week follow-up, all three treatment groups showed significant improvements compared to baseline in pain intensity (VAS; p < 0.001 for PG, BG, and CG) and lumbar mobility (Schober test; p < 0.001 for PG, BG, and CG), and functional disability (RMDQ; p < 0.001 for PG, p = 0.027 for BG, and p = 0.016 for CG). Between-group analyses at T1 revealed significant differences for RMDQ, ODI, Schober, and EQ-5D-5L. In contrast, no significant intergroup differences were observed for VAS, PPT-RS, PPT-LS, total PPT, HADS-A, HADS-D, BDI-II, or PHQ-9. Conclusions: All evaluated interventions improved clinical, functional, and psychological outcomes in patients with LBP. Prolotherapy showed a pattern of more consistent improvements, particularly in disability, spinal mobility, and health-related quality of life. These findings should be interpreted cautiously, given the non-randomized design and baseline differences between groups. Further randomized studies with larger samples and longer follow-up are warranted to confirm these results.