Abstract Background To explore and compare safety, efficiency, and health-related quality of telephone triage in out-of-hours primary care (OOH-PC) services performed by general practitioners (GPs), nurses using a computerised decision support system (CDSS), or physicians with different medical specialities. Methods Natural quasi-experimental cross-sectional study conducted in November and December 2016. We randomly selected 1294 audio-recorded telephone triage calls from two Danish OOH-PC services triaged by GPs (n = 423), nurses using CDSS (n = 430), or physicians with different medical specialities (n = 441). An assessment panel of 24 physicians used a validated assessment tool (Assessment of Quality in Telephone Triage - AQTT) to assess all telephone triage calls and measured health-related quality, safety, and efficiency of triage. Results The relative risk (RR) of poor quality was significantly lower for nurses compared to GPs in four out of ten items regarding identifying and uncovering of problems. For most items, the quality tended to be lowest for physicians with different medical specialities. Compared to calls triaged by GPs (reference), the risk of clinically relevant undertriage was significantly lower for nurses, while physicians with different medical specialties had a similar risk (GP: 7.3%, nurse: 3.7%, physician: 6.1%). The risk of clinically relevant overtriage was significantly higher for nurses (9.1%) and physicians with different medical specialities (8.2%) compared to GPs (4.3%). GPs had significantly shorter calls (mean: 2 min 57 s, SD: 105 s) than nurses (mean: 4 min 44 s, SD: 168 s). Conclusions Our explorative study indicated that nurses using CDSS performed better than GPs in telephone triage on a large number of health-related items, had a lower level of clinically relevant undertriage, but were perceived less efficient. Calls triaged by physicians with different medical specialities were perceived less safe and less efficient compared to GPs. Differences in the organisation of telephone triage may influence the distribution of workload in primary and secondary OOH services. Future research could compare the long-term outcomes following a telephone call to OOH-PC related to safety and efficiency.