Web-based multimedia annotation is a valuable tool for engaging learners with diverse materials. This study aimed to assess the effects of multimedia annotation on student performance, self-regulation, and cognitive load in an online learning environment. We developed and implemented a multimedia annotation system in an online biology class using a quasi-experimental design. The study included 64 twelfth-grade students from two classes in northern Taiwan. One class, consisting of 31 students, used the web-based multimedia annotation system (the experimental group), while the other class, with 33 students, used traditional paper-and-pencil annotations (the control group). The results showed that web-based annotation tools did not have a significant impact on students' performance or cognitive load. However, they significantly improved students' self-regulation skills, including self-observation, self-judgment, and self-response. Based on these findings, we recommend that future research ensure students have ample time to become familiar with the system, which can help reduce external cognitive load and enhance learning effectiveness. Additionally, these tools enabled teachers to better assess students' understanding and provide targeted feedback. Web-based annotation can be a valuable resource for teachers to identify and address students' learning difficulties in online environments. The study suggests several recommendations for integrating web-based multimedia annotation tools into instructional practices.