Holmium–YAG (Ho:YAG) laser lithotripsy is a multi-pulse treatment modality with stochastic effects on the fragmentation. In vitro investigation on the single-pulse-induced effects on fiber, repulsion as well as fragmentation was performed to identify potential impacts of different Ho:YAG laser pulse durations. A Ho:YAG laser system (Swiss LaserClast, EMS S.A., Nyon, Switzerland) with selectable long- or short-pulse mode was tested with regard to fiber burn back, the repulsion capacity using an underwater pendulum setup and single-pulse-induced fragmentation capacity using artificial (BEGO) stones. The laser parameters were chosen in accordance with clinical application modes (laser fiber: 365 and 200 µm; output power: 4, 6 and 10 W in different combinations of energy per pulse and repetition rate). Evaluation parameters were reduction in fiber length, pendulum deviation and topology of the crater. Using the long-pulse mode, the fiber burn back was nearly negligible, while in short-pulse mode, an increased burn back could be observed. The results of the pendulum test showed that the deviation induced by the momentum of short pulses was by factor 1.5–2 higher compared to longer pulses at identical energy per pulse settings. The ablation volumes induced by single pulses either in short-pulse or long-pulse mode did not differ significantly although different crater shapes appeared. Reduced stone repulsion and reduced laser fiber burn back with longer laser pulses may result in a more convenient handling during clinical application and thus in an improved clinical outcome of laser lithotripsy.