Objective. A group-controlled trial was conducted to explore the value of macrogene second-generation sequencing in the diagnosis, drug use, and efficacy monitoring of infectious pneumonia in premature infants. Methods. One hundred and thirty-eight premature infants with suspected infectious pneumonia treated in our hospital from March 2019 to June 2022 were selected as subjects. All patients underwent deep phlegm extraction and were randomly divided into two groups. 69 cases of control group were treated with general bacterial and fungal culture. The lavage fluid of the remaining 69 cases of observation group were detected by metagenomic next-generation sequencing (mNGS). The number of diagnosed preterm infants with infectious pneumonia was compared between the two groups, and the diagnostic value of the two methods was analyzed by the receiver operator characteristic (ROC) curve. Then, the differences in clinical efficacy, antimicrobial neonatal intensive care unit (NICU) use time, antimicrobial adjustment frequency, NICU stay time, hospital stay, and serum inflammatory factors were compared between the two groups. Results. The positive rate of mNGS pathogen detection in the lavage fluid of the observation group was 92.75% (64/69). The positive rate of the culture of the lavage fluid of the control group was 52.17% (36/69). The ROC curve analysis showed that the ROC AUC of traditional culture was 0.752 (
95
%
CI
=
0.610
-0.894), and that of mNCS was 0.934 (
95
%
CI
=
0.854
-0.999). In the observation group, there were 35 cases of bacterial infection, 20 cases of fungi, 4 cases of virus, and 5 cases of Chlamydia psittaci. In the control group, 26 cases of bacterial infection and 9 cases of fungi were detected; but viruses and other mycoplasmas could not be detected. After 2 weeks of treatment, the effective rate of the observation group was 95.31%, while that of the control group was 69.44%. The NICU use time, adjustment frequency, NICU stay time, and hospitalization time of antibiotics in the observation group were significantly less than those in the control group, and the difference was statistically significant (
P
<
0.05
). After treatment, the levels of serum interleukin-6 (IL-6), procalcitonin (PCT), and hypersensitivity-C-reactive protein (hs-CRP) in observation group were significantly higher than those in control group, and the difference was statistically significant (
P
<
0.05
). Conclusion. mNGS can improve the efficiency of clinical diagnosis of infectious pneumonia in premature infants, effectively improve the detection rate of pathogens and the clinical efficacy of premature infants. At the same time, it can also assist the clinical efficacy monitoring and adjust the treatment plan at any time.