Non-healing venous leg ulcers represent a significant healthcare problem that accounts for about $32 billion of spending in the US alone. Consequently, novel treatment strategies represent a major unmet need. The current study (part one of Phase II study [NCT04647240]) assesses the safety, tolerability and efficacy of the use of cell-free human amniotic fluid in treating venous leg ulcers that did not heal following the correction of venous reflux. Patients received cell-free amniotic fluid injections in and around the wound either weekly or biweekly over 12 weeks. Primary outcomes included safety, tolerability and efficacy assessed by complete wound closure, wound area reduction and pain reduction. Eleven patients met enrollment eligibility, and nine completed the study. Five patients achieved complete wound closure by week 12. The average percent reduction in wound area was 83.7%, and pain scores were significantly lower by the study endpoint. No difference was observed in wound healing rates between weekly or biweekly treatment, but bi-weekly treatment was associated with nominally faster recovery. Patients tolerated the treatment, and no side effects were reported. These results indicate that cell-free amniotic fluid injection is a feasible, safe and effective treatment for non-healing venous leg ulcers.