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Reduced lumbar multifidus (LM) cross-sectional area (CSA) has been linked with non-specific chronic low back pain (NCLBP); hence, lumbar stabilization exercise (LSE) is typically used as treatment. Real-time ultrasound imaging (RUSI) biofeedback can enhance performance and retention in LM activation. This study aimed to compare the effects of LSE with or without RUSI-biofeedback in patients with NCLBP. A single-blind, three-arm, parallel randomized controlled trial involving 90 participants, randomized into LSE_{RUSI-biofeedback,} LSE_{no-biofeedback}, or minimal intervention (n = 30 per group) was conducted. All interventions were administered twice weekly for 8 weeks, and outcomes (LM-CSA, pain, disability, and quality of life) were evaluated before and at 8-week and 20-week follow-ups. Compared to LSE_{no-biofeedback} and minimal intervention, LSE_{RUSI-biofeedback} demonstrated a greater increase (p < 0.05) in LM-CSA at 8 weeks (mean [95%CI] difference: 0.81 [0–1.62] and 1.79 [0.91–2.65], respectively) and at 20 weeks (1.61 [0.79–2.42] and 2.66 [1.79–2.53], respectively). LSE_{RUSI-biofeedback} showed a greater improvement (p < 0.05) in mental health scores at 8 weeks (3.63 [0.32–6.94]) and 20 weeks (7.34 [4.03–10.60]) compared to LSE_{no-biofeedback}. However, LSE_{no-biofeedback} showed a greater physical health scores at 20 weeks (− 4.89 [− 7.92 to − 1.86]) compared to LSE_{RUSI-biofeedback}. No significant differences (p > 0.05) were found between LSE_{RUSI-biofeedback} and LSE_{no-biofeedback} for pain or disability. The increase in LM-CSA significantly correlated with changes in pain (r = − 0.40 to − 0.50, p < 0.05) and disability (r = − 0.38 to − 0.42, p < 0.05). In conclusion, LSE with or without RUSI-biofeedback improved LM-CSA, pain, disability, and quality of life. However, LSE_{RUSI-biofeedback} led to a greater increase in LM-CSA. Such an increase appears to be related to changes in pain and disability. Trial registration: The study was registered in the Pan African Clinical Trials Registry (16/01/2018; PACTR201801002980602).