Background Patients with hypertrophic scars (HTS) risk reduced quality of life due to itching, pain, poor cosmesis, and restriction of movement. Despite good clinical efficacy, patients are often reluctant to undergo repeated needle injections due to pain or needle phobia. Objectives To evaluate the applicability of needle-free pneumatic jet injection (PJI) and assess changes in hypertrophic scars following a single PJI treatment with 5-fluorouracil (5-FU) and triamcinolone acetonide (TAC). Methods Twenty patients completed this blinded, randomized, controlled, split-scar trial. The intervention side of the HTS received a one-time treatment with PJIs containing a mixture of TAC + 5-FU injected at 5 mm intervals (mean 7 PJI per HTS); the control side received no treatment. Assessments were made at baseline and 4 weeks posttreatment. Outcome measures included change in (1) Vancouver Scar Scale (VSS) total score and subscores, (2) scar volume and surface area assessed by three-dimensional imaging, (3) skin microarchitecture measured by optical-coherence tomography (OCT), (4) photo-assessed scar cosmesis (0-100), (5) patient-reported pain and satisfaction (0-10), and (6) depiction of drug biodistribution after PJI. Results PJI with TAC + 5-FU significantly decreased both HTS height (-1 VSS; p = 0.01) and pliability (-1 VSS; p < 0.01) with a nonstatistically significant reduction of -1 in total VSS score (0 in control; p = 0.09). On 3D imaging, a 33% decrease in scar volume (p = 0.016) and a 37% decrease in surface area (p = 0.008) was observed. OCT indicated trends towards smoother scar surface (Ra 11.1-10.3; p = 0.61), normalized dermal microarchitecture (attenuation coefficient: 1.52-1.68; p = 0.44), and a reduction in blood flow between 9% and 17% (p = 0.50-0.79). Despite advances in VSS subscores and OCT, no improved photo-assessed cosmesis was found (-3.2 treatment vs. -1.4 control; p = 0.265). Patient-reported pain was low (2/10) and 90% of the patients that had previously received needle injections preferred PJI to needle injection. Depositions of TAC + FU were imaged reaching deep into the scar at levels corresponding to the reticular dermis. Conclusion A single PJI injection containing 5-FU and TAC can significantly improve the height and pliability of HTS. PJI is favored by the patients and may serve as a complement to conventional needle injections, especially for patients with needle phobia.