Background Stroke is a leading cause of death and disability with effective treatment, including thrombolysis or thrombectomy, being time-critical for favourable outcomes. While door-to-needle time in hospital has been optimized for many years, little is known about the ambulance on-scene time (OST). OST has been reported to account for 44% of total alarm-to-door time, thereby being a major time component. We aimed to analyse ambulance OST in stroke patients eligible for thrombolysis and identify potential areas of time optimization. Methods A study-specific registration form was developed to record detailed information about OST consumption in cases where the Emergency Medical Services (EMS) suspected a stroke from July 2014–May 2015. Registration forms were completed by ambulance personnel and included details on estimated time spent: 1) localising patient, 2) clinical examination, 3) consulting with the on-call neurologist, 4) mobilising patient to the ambulance, 5) treatment in ambulance before departure. Additionally, estimated total OST was noted. For patients found eligible for further evaluation at a stroke centre, time points were analysed using multivariate Poisson regressions. Results A total of 520 cases were included. The median OST was 21 min (Interquartile Range (IQR) 16–27). Time consumption was significantly lower (17 vs 21 min, p = 0.0015) when electrocardiography (ECG) was obtained in-hospital instead of on-scene, when intravenous (IV) access was established during transportation instead of before transportation (17 vs 21 min, p < 0.0001), and when the quality of communication with the stroke centres was rated as “good” as opposed to “acceptable/poor” (21 vs 23 min, p = 0.014). Neither the presence of relatives nor ambulance trainees had a significant effect on OST. Conclusions In-hospital ECG recording and IV cannulation during transport were found to reduce OST, while “acceptable/poor” communication was found to prolong OST relative to “good” communication. These components of pre-hospital stroke management represent potential opportunities for lowering OST with relatively simple changes, which could ultimately lead to earlier treatment and better patient outcome.