Objective: To analyse the influence of root canal instrumentation and obturation techniques on intra-operative pain experienced by patients during endodontic therapy. Method and Materials: A descriptive cross-sectional study was carried out in Ponferrada and Sevilla, Spain, including 80 patients (46 men and 34 women), with ages ranged from 10 to 74 years, randomly recruited. Patient gender and age, affected tooth, pulpal diagnosis, periapical status, previous NSAID or antibiotic (AB) treatment, and root canal instrumentation and obturation techniques were recorded. After root canal treatment (RCT), patients completed a 10-cm visual analogue scale (VAS) that ranked the level of pain. Results were analysed statistically using the Chi-square and ANOVA tests and logistic regression analysis. Results: The mean pain level during root canal treatment was 2.9 ± 3.0 (median = 2) in a VAS between 0 and 10. Forty percent of patients experienced no pain. Gender, age, arch, previous NSAIDs or AB treatment and anaesthetic type did not influence significantly the pain level (p > 0.05). Pain during root canal treatment was significantly greater in molar teeth (OR = 10.1; 95% C.I. = 1.6 - 63.5; p = 0.013). Root canal instrumentation and obturation techniques did not affect significantly patient’s pain during root canal treatment (p > 0.05). Conclusion: Patients feel more pain when RCT is carried out on molar teeth. The root canal instrumentation and obturation techniques do not affect significantly the patients’ pain during RCT.