Background Chronic nonspecific low back pain (LBP) is one of the common health issues. Hamstring tightness contributes to the development of LBP. This study aimed to investigate the acute and short-term effects of deep dry needling (DN) in patients with chronic nonspecific LBP and hamstring muscle tightness. Methods A single-group pretest-posttest clinical study design was followed. The outcome measures were the visual analog scale (VAS), passive knee extension (PKE) test, finger-floor distance (FFD) test, and functional rating index (FRI). Patients underwent one session of deep DN of three points on both hamstring muscles, each point for one minute. Patients were assessed before (T0), immediately after (T1), and one week after DN (T2). The FRI was assessed at T0 and T2. Results Ten women with a mean age of 21.1 years (SD = 1.6) participated in the study. Significant large effect sizes in VAS pain reduction (d = 1.25) and PKE hamstring tightness were obtained (hamstring: right, d = 0.82; left, d = 0.88) at T2. Medium effect sizes were obtained for FFD (d = 0.45) and FRI (d = 0.72) at T2. Conclusion A single session of deep DN improved pain and function and increased hamstring flexibility. This pilot study supports the use of DN in patients with LBP and hamstring tightness; however, future research with a rigorous study design of randomized controlled trial is required to confirm the findings. This trial is registered with IRCT20180511039612N1.