Background: This study aimed to assess the attitude of Jordanian healthcare workers (HCWs) toward modern assisted reproductive technologies (ARTs), including gamete donation and sex selection. Design and Methods: The HCWs from three municipalities in Jordan were randomly selected to participate in a self-administered, cross-sectional survey. The study settings included hospitals and clinics in the public and private health sectors. Logistic regression analysis was used to examine the association of demographic characteristics and fertility experience with the attitude toward modern ARTs. Results: A total of 382 HCWs were included in the study. Most participants did not support the donation of fertilized or unfertilized eggs for any purpose (86.4% and 75.4%, respectively). Overall, only 39.3% and 42.9% of the respondents supported the use of in vitro fertilization and intrauterine insemination for sex selection, respectively. Religious considerations were the main reason (71.1%) for the negative attitude toward ARTs. However, more supportive attitude was observed among male participants and medical doctors. Logistic regression analysis showed that none of the variables were significantly associated with the attitude toward sex selection and gamete donation. Conclusion: The use of ARTs was generally more acceptable for sex selection than for gamete donation. The attitude of HCWs was primarily driven by religious beliefs, which typically shape the cultural values and the acceptance of such technologies. Additionally, sex and profession seem to play a significant role in shaping the attitude of HCWs toward ARTs.