Latent fingermarks (FMs) present unique, and sometimes the only, evidence found at a crime scene. Several factors affect their quality, including deposition pressure (DP). Its effect on FM size and quality, and on STR amplification success rate, is an emerging area of interest in forensic science. This study examined 540 FM samples, each consisting of index, middle and ring fingers, deposited by 30 donors on glass, polythene (PE) and paper under a range of weights from 0.1 to 10 kg. Both length and width of FMs increased with the increasing DP. FMs deposited under lower (≤0.5 kg) DPs varied in size (p < 0.01), while those deposited at higher (≥3 kg) DPs were more consistent. FM quality on glass and PE, as determined by the AFIS minutiae count and by a fingerprint examiner on a scale from 0 to 4, improved with the increasing DP, but it deteriorated on PE at DP of 10 kg. FM quality on paper continued to improve from DP of 1 kg up to the maximum DP of 10 kg. The effect DP has on the efficacy of DNA profiling from latent FMs was significant as shown by an increase in the DNA amount recovered, the number of amplified loci per FM sample, and the number of forensically useful DNA profiles (defined here as those with ≥8 full STR loci detected) as DP increased. This effect was most pronounced with PE (R = 0.98) and paper (R = 0.96). Altogether, the success rate of DNA profiling varied from 16.3% in FMs deposited on paper to 21.2% and 22.5% of those on PE and glass. The highest number of useful DNA profiles was obtained from glass under DP of 10 kg. Forensically useful FMs obtained at low (≤1 kg) DP from all three substrates significantly outnumbered that of STR profiles, while an opposite, though less pronounced trend, was observed at high (≥3 kg) DP on PE and paper. Application of the simple device for collecting of FMs under controlled pressure designed for this study, and the palm-up mode of FM deposition as described, allowed us to eliminate the undesirable effect of the hand self-weight and to objectively assess the actual effect of increasing DP on FM size and quality, as well as on the efficacy of DNA profiling.